内质网应激效应分子CHOP驱动肾小管上皮细胞HMGB1核浆迁移在肾间质纤维化中的作用和机制研究

The initiation of renal interstitial fibrosis is strongly associated with renal tubular epithelial cells injury and apoptosis. However, the knowledge of involved mechanism and signal pathway is still limited. Our previous studies showed that endoplasmic reticulum stress effector molecule CHOP (C/EBP homologous protein) promotes renal interstitial fibrosis via up-regulating apoptosis and the secretion of proinflammatory and profibrotic factors in renal tubular epithelial cells, while we also observed the nuclear-cytoplasmic translocation and extracellular release of DAMP (Damage-Associated Molecular Pattern) molecule HMGB1 in renal interstitial fibrosis animal model. Therefore, we hypothesized that the crucial mechanism initiating renal interstitial fibrosis is: renal tubular epithelial cells injury leads to endoplasmic reticulum stress, followed by its effector molecule CHOP inciting the nuclear protein HMGB1 nuclear-cytoplasmic translocation and extracellular release, and then extracellular HMGB1 induces cascade inflammation to enhance renal interstitial fibrosis. To test this hypothesis, we will use CHOP knockout(-/-) mice, CHOP small interfering RNA and anti-HMGB1 neutralizing antibody in unilateral ureteral obstruction (UUO) model and TGF-β1 stimulated cells, to investigate the nuclear-cytoplasmic translocation of HMGB1 and the expression of its regulatory protein PPARγ in renal tubular epithelial cells , and identify that the initiation of renal fibrosis is mediated by CHOP via PPARγ regulating nuclear-cytoplasmic translocation of HMGB1, and finally provide a potential therapeutic strategy for chronic renal fibrosis.

肾间质纤维化(renal interstitial fibrosis, RIF)启动机制不明，是目前制约肾脏纤维化研究的瓶颈。肾小管上皮细胞损伤和凋亡被认为参与RIF的发生，但其触发RIF的具体信号途径至今仍缺乏清晰认识。我们前期研究发现内质网应激效应分子CHOP介导肾小管上皮细胞凋亡可能是RIF的始动因素，在RIF的动物模型中伴随HMGB1的核浆迁移和胞外释放。因此我们推测损伤导致内质网应激效应分子CHOP 驱动“损伤相关分子模式”分子HMGB1迅速从胞核迁移到胞浆胞外，诱导“瀑布般”级联炎症反应，释放促纤因子，是RIF启动的核心环节。本项目拟通过CHOP-/-小鼠、CHOP SiRNA和抗HMGB1中和抗体等，研究HMGB1核浆迁移及其调控蛋白PPARγ的变化，探讨CHOP通过PPARγ驱动肾小管上皮细胞HMGB1核浆迁移，启动RIF的假说，为RIF的防治提供新的理论依据和治疗靶点。

急性肾损伤(Acute kidney injury)的机制不明确，目前除了血液透析外，没有明确有效的治疗急性肾损伤的方法，且急性肾损伤与慢性肾脏病密切相关。肾小管上皮细胞损伤和死亡是急性肾损伤发生发展的关键环节，肾小管上皮细胞的各种死亡方式途径至今仍缺乏清晰认识。我们研究发现肾小管高迁移率族蛋白 1(HMGB1)在肾脏疾病中的作用广泛，肾小管上皮细胞核浆迁移参与了肾脏疾病，本项目通过CRISPR/Cas9基因编辑技术构建肾小管特异性的HMGB1敲除鼠，在缺血诱导的急性肾损伤中，肾小管特异性HMGB1敲除鼠损伤明显减轻，炎性细胞浸润减少，鉴于“损伤相关分子模式”分子HMGB1参与多种细胞死亡方式，我们发现HMGB1的敲除只影响了铁死亡ferroptosis，而与程序性死亡无明显关系。由于HMGB1表达部位不同，功能迥异，我们通过HMGB1重组蛋白的使用发现，胞外的HMGB1并不影响其敲除所致的肾损伤减轻。另外，我们发现铁死亡的表达与临床肾功能表达具有相关性，因此抑制铁死亡可能是治疗急性肾损伤最重要的靶点。